Flaviviridae family comprises three genuses of Flavivirus, Pestivirus, and Hepacivirus. Genus Flavivirus genomes consist of a linear, single-stranded, positive sense RNA having a total genome range of 10 to 11 kilobase pairs, which forms a structure of 5′-stuctural genes-nonstructural genes-3′. The 3′ terminus of Flavivirus genomes is not polyadenylated, and the 5′ end has a methylated nucleotide cap (allowing for translation) or a genome-linked protein. Virus structural genes on 5′-end occupy one-fourth of the whole genome, which comprises C, prM, and E genes, and non-structural genes occupy the remaining parts of the genome. The virions appear roughly as spheres, 40-65 nm in diameter comprising three structural proteins: truncated envelope protein (E protein), membrane protein (M protein), and capsid protein (C protein). The number of viral non-structural proteins existing in Flavivirus-infected cells is not quite sure. However there are at least three non-structural proteins identified in Flavivirus-infected cells and those proteins are highly related to viral RNA replication, wherein at least one protein has a proteinase function. Truncated envelope protein (E protein) is a viral agglutinin that will allow infected cells to adhere hemoglobin. The 5′-UTR (un-translated region) is a highly conserved region in viral genome and an important part in the initiation or control of protein translation (Thurner C., et al., J Gen Virol. 2004 May; 85(Pt 5): 1113-24; Henchal E. A. and Putnak J. R. Clin Microbiol Rev. 1990 October; 3(4): 376-396; Chambers T. J. et al., Annu Rev Microbiol. 1990; 44:649-88.)
Genus Flavivirus comprises at least 65 species being either direct pathogens for humans or zoonosis. Except for the hepatitis C virus, which is spread by body fluid contact, the media for other viruses are arthropods such as mosquitoes or ticks. There are three clinical syndromes observed for Flavivirus infection including: central neuron disease caused by Flavivirus such as St. Louis encephalitis, murray valley encephalitis, Japanese encephalitis, and the like; systematic disorder caused by yellow fever virus infecting organs; and serious muscle disorders (such as acute flaccid paralysis (AFP), peripheral demyelinating process (Guillain-Barré Syndrome (GBS), anterior myelitis and the like), and hemorrhagic fever caused by West Nile virus, dengue fever virus, and the like). According to estimation from the World Health Organization (WHO), just for the dengue fever virus, about 2 million infections result every year globally. Over two thousand cases including 77 deaths have been reported for the West Nile virus infection from January to November of 2004 according to statistical data published by the CDC (USA) dated on Nov. 8, 2004. Flavivirus infection has become a major issue on worldwide epidemiology.
The infection of Flavivirus needs to be confirmed by virus isolation and serological identification; wherein yellow fever virus, dengue virus, and some encephalitis cases caused by ticks can be isolated from a blood sample. However, the serological identification of virus is not useful for therapy due to the low crossover antibody protection. Further, a vaccine for Japanese encephalitis has been widely used for years; however, because said vaccine is a live attenuated virus vaccine, there's a lime limitation for titer maintenance. In the past, people who had received vaccination used to obtain a natural boost due to frequently being bilten by mosquitoes. The chance to get a natural boost is lower today due to improved living conditions. Therefore, there are still some Japanese encephalitis infected cases reported from vaccinated adults. Currently, antiviral drugs are available for the treatment of HIV, herpes virus (pathogens for various diseases such as Herpes labialis or encephalitis), and hepatitis B or C viruses (both can result in liver cancer). There is no available or anticipated drug for the clinical treatment of Flavivirus infection, and syndrome supportive therapy has been applied in most cases. Therefore, it is urgently needed to have a simple and rapid method for screening natural or synthetic compounds that might be useful in preventing the Flavivirus infection.
The currently used screening method for anti-virus drugs comprises: infecting cells with virus, culturing the infected cells in a culture medium, adding possible compounds to the culture medium, and further examining the compounds to see which can decrease viral numbers. The above method is not economically efficient in that the laboratory staff needs to frequently repeat the step of infecting cells with virus. In addition, compounds selected by the above screening method are not guaranteed to inhibit intercellular viral protein expression. There are still some doubts as to whether or not the selected compounds can enter cells and the expressed cytoxicity on cells.